The BIF offers a one-week course in Microtechnique taught by Dr. Denise Schichnes. It is a hands-on lab class where you will learn the fundamentals of making microscope slide preparations for research.
While the student will be given a short workbook on the topic, you may consider this text by Ruzin.
Plant Microtechnique and Microscopy
OXFORD UNIVERSITY PRESS
ISBN 0-19-508956-1
BY
STEVEN E. RUZIN
BIOLOGICAL IMAGING FACILITY
COLLEGE OF NATURAL RESOURCES
UNIVERSITY OF CALIFORNIA, BERKELEY
Plant microtechnique – the preparation of microscope slides from living material – has made a resurgence due, in part, to the necessity of molecular biologists to visualize a gene or gene product in the context of the whole plant. Molecular biologists are no longer content with knowing that a gene is just being expressed; they need to know where in the organism and when during its developmental program that gene is doing what it does. This is where microtechnique comes into play. The “old fashioned” techniques of microscopy and paraffin sectioning have become indispensable. The fact that the instruction manuals are dated has resulted in this manual. This Laboratory Manual in Plant Microtechnique started from a series of handouts from my course at Berkeley in Plant Microtechnique. In the six years it took to write this text, I added background, techniques, protocols, and tables, in an effort to include most of the information a student in developmental biology requires for an anatomical and cytological investigation of plants. Finally, although the text is geared toward plant biology, it is applicable to animal biology as well.
Contents
Introduction
Abbreviations
1 Quick Start
- Paraffin embedding &endash; Histology or in situ hybridization
- Paraffin embedding &endash; Microwave method
- Glycol methacrylate embedding &endash; Histology
- Butyl/methyl methacrylate embedding &endash; In situ hybridization or immunolocalization
- Steedman’s wax embedding &endash; Immunolocalization
- Chromosome squashes &endash; Maize meiocytes
- Hematoxylin staining &endash; Microwave technique
- Adjusting Köhler illumination
2 Microscopy
- Magnifiers and microscopes
- Compound microscopes
- Principles of specimen illumination
- Imparting contrast using inherent differences in index of refraction within the specimen
- Phase Contrast Microscopy
- Hoffman Modulation Contrast Microscopy
- Confocal and wide-field deconvolution microscopy
- Obtaining microscopic measurements
3 Chemical Fixation
- Types of chemical fixatives
- The quality of fixation
- Tissue storage
- The mechanics of fixation
- Coagulating Fixatives
- Noncoagulating fixatives
- Fixatives for Cytology
- Recommendations for fixing plant tissues
4 Tissue dehydration
- General protocol
- Dehydration using a graded solvent series &endash; Ethanol, acetone
- Rapid dehydration
5 Infiltrating and embedding tissues
- Paraffin infiltrating and embedding
- Polyethylene glycol and derivatives &endash; Infiltration and embedding
- Plastic infiltrating and Embedding
6 Sectioning and Mounting
- General steps
- Mounting blocks to microtome stubs
- Factors influencing the quality of sectioning
- The microtome knife
- Setting up the microtome
- Sectioning methods
- Mounting sections to glass slides
7 Staining
- Chemical classification of dyes
- Dye chemistry
- Microtechnique stains
- General staining procedures
- Staining equipment
- Selected staining methods for plant microtechnique
- Staining epoxy-embedded tissues
- Reactive dyes
- Cytogenetic techniques with squashed material
- Cytogenetic stains for sectioned material
- Staining pollen tubes
- Mounting the coverslip
8 Alternate methods of microtomy
- Free-hand sections
- Vibratome
- Freezing microtome (cryotome, cryostat)
- Sliding microtome
9 Special Methods
- Tissue clearing
- Macerating woody tissues
- Macerating gymnosperm leaves for epidermal peels
- Macerating nonwoody tissues
- Sectioning carbonized wood samples
- Microincineration
- Bleaching tissues
- Preserving color in whole mounted specimens
- Repairing broken microscope slides
10 Microtechnique notes and problem solving
- Tissue collection
- Fixation
- Fixatives for algae
- Tannins
- Hard materials
- Sliding microtome
- Clearing
- Dehydration and infiltration
- Embedding
- Microtomy
- Free hand sections
- Staining
11 Histochemistry and cytochemistry
- The relationship of the probe to the target
- Cytochemical localization of cell components
- Cytochemical localization of enzymes
- Cytochemical fluorescence microscopy
- Fluorescent dyes for specific applications
- Intrinsic fluorescence
- Background fluorescence and photobleaching
12 Localization of molecular targets in tissues
- Immunolocalization
- Example protocols for immunolocalization
- Tissue printing to detect proteins and RNA in plant tissue
- In situ hybridization
- Autoradiography and Detecting incorporated radioactive elements in tissue sections
- Whole-mount in situ hybridization
- Tyramide signal amplification
- TUNEL Assay for detecting DNA degradation and programmed cell death
- Fluorescence in situ hybridization
Appendices
- I Toxics
- II Buffers
- III General Information and Common Calculations
- IV The Mercury Arc Lamp in Fluorescence Microscopy
- V Manufacturers and Vendors
- VI Optics
Bibliography
Index
Steven Ruzin, Ph.D.
CNR Biological Imaging Facility
Department of Plant and Microbial Biology
University of California
Berkeley, CA 94720-3102